Rebridging disulphides: site-specific PEGylation by sequential bis-alkylation

Site-specific PEGylation reagents have been developed that undergo thiol-specific bis-alkylation with the two cysteine sulphur atoms from a native accessible disulphide in proteins. The process for this approach of site-specific PEGylation involves two steps: (1) disulphide reduction to release the two thiols and (2) bis-alkylation of the PEG reagent to the two sulphur atoms to give a three-carbon bridge to which PEG is covalently attached. Mechanistically, the conjugation is thought to occur by a sequential, interactive bis-alkylation that requires functionalised PEG reagents that have a α, β-unsaturated β′-mono-sulphone moiety. Competitive reactions can be effectively suppressed to achieve high yield PEGylation with a stoichiometric equivalence of the reagent. The reagents are easily prepared and precursor forms of our PEG reagents can be used to control the rate of formation of the reactive PEG mono-sulphone in situ that undergoes conjugation with the protein. Purification is often a simple process where un PEGylated protein can be easily recycled to further increase yields. Many classes of therapeutically relevant proteins possess accessible native disulphides. Our studies have shown that peptides, proteins, enzymes and antibody fragments can be site-specifically PEGylated by bis-alkylation using a native, accessible disulphide.